Conventional chemotherapy is effective initially in controlling and reversing tumor growth. However, residual cancers will invariably re-grow. Our recent data from paired human breast cancer samples indicates that standard therapy eliminates dividing daughter cells while enriching the residual tumor for CD44?/low putative breast "cancer stem cells" (CSCs) that have the ability to self-renew in mammosphere (MS) cultures, and to give rise to tumors upon xenograft transplantation. We have identified a cancer stem cell signature within these CD44?/low, MS-forming cells that features Stat3. Using virtual ligand screening (VLS), we developed lead chemical probes that target the Src homology (SH) 2 domain of Stat3 and that competitively and selectively inhibited Stat3 activation. 3-D pharmacophore analysis was performed using the most active lead compound (Cpd188) and a Life Chemicals compound library;the top scoring compounds identified were tested for inhibition of Stat3 binding to its phosphopeptide ligand by surface plasmon resonance (SPR). All but six of 39 compounds tested have measurable IC50s, with 17 having IC50 values equal to or less than Cpd188. One compound (Cpd188-15) has an IC50 value in MS formation inhibition assays that is one log lower than Cpd188. We have developed 2 highly focused SPECIFIC AIMS to directly test the hypothesis that small- molecule inhibitors of Stat3 can specifically target breast cancer stem cells. AIM 1. To develop third (3rd) generation Stat3 chemical probes with increased binding affinity and inhibitory activity. We propose using the most active 2nd generation probe, Cpd188-15, as a scaffold for making 3rd generation probes. Modifications planned are based on the results of a structure-activity relationship (SAR) analysis performed on 2nd generation probes and center around the straightforward synthesis of sulfamides from panels of sulfonyl chlorides and amides. Each novel sulfamide compound will be examined in the SPR assay, in a high throughput fluorescence microscopy (HTFM) assay that will test inhibition of IL-6-stimulated cytoplasmic-to-nuclear translocation and for their selectivity for Stat3 vs. Stat1. AIM 2. To determine whether suppression of the Stat3 pathway can improve existing cancer therapies in preclinical MS assays and human xenograft models. We will determine whether Stat3 inhibition by the most active 3rd generation probes will improve efficacy of conventional therapy in vitro and in vivo, using MS formation inhibition assays and human breast cancer xenograft models. In addition to establishing the contribution of Stat3 to breast cancer stem cell survival and self-renewal, these preclinical studies may inform the design of future clinical trials and determine whether suppressing stem cell self-renewal and treatment resistance pathways can improve existing breast cancer therapies in patients. PUBLIC HEALTH RELEVANCE: A small proportion of breast cancer cells fail to respond to the usual treatments and cause relapsing disease. This proposal outlines chemical and biological studies designed to develop small molecule probes that target these therapy-resistant cells. If these studies are successful, these small molecule probes may be combined with current treatments for breast cancer thereby preventing cancer relapse and resulting in a cure.